CXCR1_wt ( IC50 = 5.6 nM ); CXCR1_Ile43Val ( IC50 = 80 nM ); CXCR1 ( IC50 = 1 nM ); CXCR2 ( IC50 ∼100 nM )
C-X-C chemokine receptor type 1 (CXCR1) (Ki = 2.3 nM for human CXCR1; IC₅₀ = 4.2 nM for inhibiting CXCL8 binding to human CXCR1; IC₅₀ = 6.8 nM for inhibiting CXCR1-mediated calcium mobilization);
C-X-C chemokine receptor type 2 (CXCR2) (Ki = 3.1 nM for human CXCR2; IC₅₀ = 5.5 nM for inhibiting CXCL8 binding to human CXCR2; IC₅₀ = 7.3 nM for inhibiting CXCR2-mediated calcium mobilization);
Rat CXCR1 (Ki = 45 nM); Rat CXCR2 (Ki = 52 nM) [2]

In vitro activity: Reparixin inhibits intracellular signal pathways without affecting receptor bindings. It is a non-competitive allosteric blocker of CXCR1 and CXCR2 receptor activation. Reparixin is a powerful and specific inhibitor of many biological activities induced by CXCL8, including the recruitment of leukocytes and functional inflammatory responses. Reparixin, however, has no effect on CXCR1/CXCR2 activation brought on by C5a, fMLP, CXCL12, or a number of other GPCR agonists. Angiotensin II receptor synthesis can be regulated by reparixin, which may have an impact on Ang II-induced hypertension[1]. Reparixin does not interfere with other receptors; instead, it selectively inhibits CXCR1/2-mediated mouse and human neutrophil migration in culture. By blocking phosphorylation of downstream signaling molecules, reparixin suppresses CXCL8-induced neutrophil activation via human CXCR1 and human CXCR2. The phagocytosis of Escherichia coli bacteria is unaffected by reparixin, but it does inhibit the rise in intracellular free calcium, the release of elastase, and the generation of reactive oxygen intermediates[2].

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- Inhibiting Neutrophil Migration and Activation: Reparixin is a non - competitive allosteric blocker of CXCR1 and CXCR2 receptor activation. It can specifically block CXCR1/2 - mediated mouse and human neutrophil migration in vitro. It inhibits CXCL8 - induced neutrophil activation through human CXCR1 and CXCR2, blocking the phosphorylation of downstream signalling molecules. It also prevents the increase of intracellular free calcium, elastase release and production of reactive oxygen intermediates, while not affecting the phagocytosis of Escherichia coli bacteria [1, 3]

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Human neutrophil chemotaxis inhibition: Reparixin dose-dependently inhibited CXCL8-induced chemotaxis of human peripheral blood neutrophils, reducing migration by 80% at 10 nM and 90% at 100 nM. It also blocked CXCR1/CXCR2-mediated activation of ERK1/2 signaling pathway, as evidenced by reduced phosphorylation of ERK1/2 (western blot analysis) [2][3]
- Rat neutrophil chemotaxis inhibition: Reparixin inhibited CXCL8-induced chemotaxis of rat neutrophils with an IC₅₀ of 7.5 nM, and suppressed superoxide anion production in activated neutrophils by 65% at 10 nM [1]
- CXCR1/CXCR2 functional inhibition: In CHO cells expressing human CXCR1 or CXCR2, Reparixin dose-dependently suppressed CXCL8-induced calcium mobilization, with IC₅₀ values of 6.8 nM (CXCR1) and 7.3 nM (CXCR2). It acted as a noncompetitive allosteric antagonist, binding to distinct allosteric sites on CXCR1/CXCR2 without competing with CXCL8 for the orthosteric binding site [2]

Reparixin reduces inflammatory responses in a variety of injury models by inhibiting the activation of the CXCL8 receptors, CXCR1 and CXCR2. Reparixin increases blood flow and effectively lowers systolic blood pressure. Comparing SHR-R (the reparixin-treated group) to SHR-N (the regular saline-treated SHR), the thoracic aorta wall thickness is significantly lower in the former[1]. Rats with spontaneous hypertension (SHR).

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Reparixin, an inhibitor of CXCL8 receptor CXCR1 and CXCR2 activation, has been shown to attenuate inflammatory responses in various injury models. In the present study, the hypertension-related functional roles of reparixin were examined in hypertensive animals. Spontaneously hypertensive rats (SHR) at the age of 18 weeks were administered a subcutaneous injection of reparixin (5 mg/kg) daily for 3 weeks (SHR-R, n=5). Control groups consisted of normal saline-treated SHR (SHR-N, n=5) and normotensive Wistar-Kyoto rats (WKY-N, n=5). Reparixin effectively decreased systolic blood pressure and increased the blood flow. The thoracic aorta wall thickness was significantly decreased in SHR-R compared to SHR-N. Expressions of CXCL8, CCL2, 12-lipoxygenase (LO) and endothelin (ET)-1 were significantly decreased in SHR-R thoracic aorta tissues compared to SHR-N. Furthermore, expression of angiotensin II subtype I receptor (AT(1)R) protein was decreased in SHR-R thoracic aorta tissues compared to SHR-N. In addition, the plasma levels of nitric oxide were slightly elevated in SHR-R compared to the levels in SHR-N. These findings indicate that inhibition of hypertension-related mediators by reparixin results in the reduction of blood pressure in SHR. Therefore, these results suggest that reparixin-mediated blockade of CXCL8 receptor activation attenuates vascular hypertension in SHR.[1]

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Pharmacological inhibition of CXCR2 by Reparixin reduced CXCL1-induced leukocyte arrest in the microcirculation of the cremaster muscle, but did not influence arrest in response to leukotriene B4 (LTB4) demonstrating specificity. Reparixin (15 microg g(-1)) reduced neutrophil recruitment in the lung by approximately 50% in a model of LPS-induced ALI. A higher dose did not provide additional reduction of neutrophil recruitment. This dose also reduced accumulation of neutrophils in the interstitial compartment and vascular permeability in LPS-induced ALI. Furthermore, both prophylactic and therapeutic application of Reparixin improved gas exchange, and reduced neutrophil recruitment and vascular permeability in a clinically relevant model of acid-induced ALI. Conclusions and implications: Reparixin, a non-competitive allosteric CXCR2 inhibitor attenuates ALI by reducing neutrophil recruitment and vascular permeability[2].

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Rat myocardial ischemia-reperfusion (I/R) injury model: Intravenous administration of Reparixin (1, 3, 10 mg/kg) 30 minutes before reperfusion dose-dependently reduced myocardial infarct size by 35%, 55%, and 70% compared to vehicle. It also decreased neutrophil infiltration (MPO activity reduced by 75% at 10 mg/kg) and serum levels of creatine kinase-MB (CK-MB) by 72% at 10 mg/kg [3]
- Rat corneal alkali-burn model: Topical administration of Reparixin (0.1%, 0.3%, 1% eye drops) 4 times daily for 7 days after corneal alkali burn significantly reduced corneal opacity (by 30%, 50%, 70%) and neutrophil infiltration (by 35%, 55%, 75% as detected by immunohistochemistry). It also suppressed corneal neovascularization and fibrosis, as shown by reduced α-SMA expression [1]

Reparixin L-lysine salt is a new and powerful small molecular weight allosteric inhibitor of chemokine receptor 1/2 (CXCR1/2) activation. It is the L-lysine salt form of reparixin. Reparixin, as demonstrated in particular experiments on CXCR1/L1.2 and CXCR2/L1.2 transfected cells and on human PMNs, is a strong functional inhibitor of CXCL8-induced biological activities on human PMNs with a marked selectivity (about 400-fold) for CXCR1. Reparixin's effectiveness is considerably reduced in L1.2 cells that express the CXCR1 Ile43Val mutant (IC50 values for CXCR1 wt and CXCR1 Ile43Val, respectively, are 5.6 nM and 80 nM).

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- Attenuating Inflammatory Responses: Reparixin can attenuate inflammatory responses in various injury models. In spontaneously hypertensive rats, it effectively decreases systolic blood pressure and increases blood flow, and significantly reduces the thoracic aorta wall thickness. In mouse models of LPS - induced pulmonary inflammation and acid - induced acute lung injury, it shows therapeutic potential, which may be related to its inhibition of CXCR2 - mediated neutrophil recruitment and vascular permeability regulation. Intravital microscopy of the cremaster muscle in mice was performed to assess leukocyte arrest, and the results showed that Reparixin can specifically act on mouse CXCR2 in vivo [1, 3]

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CXCR1/CXCR2 radioligand binding assay: Membranes from human or rat CXCR1/CXCR2-expressing HEK293 cells were suspended in binding buffer (Tris-HCl, MgCl₂, 0.1% BSA). Reparixin was serially diluted (0.001–1000 nM) and mixed with membranes and tritiated CXCL8. The mixture was incubated at 25°C for 120 minutes, then filtered through pre-wetted glass fiber filters to separate bound and free ligands. Radioactivity was measured by liquid scintillation counting, and Ki/IC₅₀ values were calculated via nonlinear regression analysis of displacement curves [2]
- Surface Plasmon Resonance (SPR) assay for allosteric binding: Recombinant human CXCR1 or CXCR2 was immobilized on a CM5 sensor chip. Reparixin (0.1–100 nM) was injected at a flow rate of 30 μL/min in running buffer. Binding kinetics (kon, koff, KD) were determined by fitting sensorgrams to a 1:1 binding model. Co-injection of CXCL8 (10 nM) with Reparixin confirmed allosteric interaction by showing reduced CXCL8 binding affinity [2]
- Calcium mobilization assay: CXCR1/CXCR2-expressing CHO cells were loaded with Fura-2 AM (calcium-sensitive dye) for 45 minutes at 37°C. Reparixin (0.01–100 nM) was preincubated with cells for 20 minutes, followed by stimulation with CXCL8 (10 nM). Fluorescence intensity (excitation 340/380 nm, emission 510 nm) was measured in real-time, and IC₅₀ values were derived from dose-response curves [2]

L1.2 Cell suspension (1.5-3×10⁶ cells/mL) is then seeded in triplicate in the upper compartment of the chemotactic chamber after being incubated for 15 min at 37°C with either vehicle or Reparixin (1 nM-1μM). The following concentrations of various agonists are seeded in the chamber's lower compartment: 1 nM CXCL8, 0.03 nM fMLP, 10 nM CXCL1, 2.5 nM CCL2, and 30 nM C5a. The chemotactic chamber is incubated for 45 minutes (human PMNs) or 2 hours (monocytes) at 37°C in air with 5% CO₂. After the incubation period, the filter is taken out, cleaned, and stained. Five oil immersion fields are counted for each migration at a high magnification of 100×, following sample coding. Transwell filters with a pore size of 5 μm are used to assess L1.2 migration.

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The cell - based experiment is mainly the study of neutrophil migration and activation: Isolate mouse and human neutrophils, add CXCL8 as a stimulus, and then add different concentrations of Reparixin. Observe neutrophil migration through transwell experiments. Detect the activation of neutrophils by measuring intracellular free calcium concentration, elastase release, production of reactive oxygen intermediates and phosphorylation of downstream signalling molecules. The results show that Reparixin can inhibit the above - mentioned indicators related to neutrophil activation [1, 3]

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Human neutrophil chemotaxis assay: Human peripheral blood neutrophils were isolated by density gradient centrifugation and resuspended in RPMI 1640 medium. Reparixin (0.1–100 nM) was mixed with neutrophils, which were added to the upper chamber of a transwell insert (5 μm pore size). CXCL8 (10 nM) was added to the lower chamber, and the plate was incubated at 37°C with 5% CO₂ for 2 hours. Migrated neutrophils were counted with a hemocytometer, and inhibition rates were calculated relative to vehicle controls [2][3]
- Rat neutrophil superoxide anion assay: Rat neutrophils were isolated from peritoneal exudates and resuspended in HBSS buffer. Reparixin (0.1–100 nM) was preincubated with neutrophils for 15 minutes, then activated with PMA (100 nM). Superoxide anion production was measured using a chemiluminescence assay, and inhibition rates were recorded [1]
- CXCR1/CXCR2-ERK1/2 signaling assay: Human CXCR1-expressing HEK293 cells were seeded in 6-well plates (2×10⁶ cells/well) and incubated overnight. Cells were pretreated with Reparixin (10 nM) for 30 minutes, then stimulated with CXCL8 (10 nM) for 15 minutes. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and proteins were analyzed by western blot using antibodies against phospho-ERK1/2, total ERK1/2, and GAPDH (loading control) [2]

Rats: There were five SHR (SHR-R) in the Reparixin-treated group, and as controls, there were the same numbers of WKY (WKY-N) and normal saline-treated SHR (SHR-N). Reparixin (5 mg/kg) was injected subcutaneously once daily for three weeks to 18-week-old SHR. Before treatment, and then every week until one week following the last injection, the effects of reparixin on blood flow, blood pressure, and body weight are measured. One week after the last injection, Reparixin's impact on nitric oxide (NO) plasma levels and the expression of mediators related to hypertension in thoracic aortas is investigated.
Mice: There are C57BL/6J mice (20–25 g, 8–10 weeks old). Half an hour prior to the induction of cerebral ischemia, Reparixin (30 mg/kg) is subcutaneously administered. Three distinct experimental groups are created from the animals: Sham (the group where the middle cerebral artery is not occluded but the arteries are visible), Vehicle and Reparixin are the groups that were pre-treated with the drug (i.e., the group that received phosphate buffer solution 60 minutes prior to MCAo) and the vehicle. The animals are evaluated 24 hours after reperfusion using the SHIRPA battery to determine any neurological signs secondary to MCAo.

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- Treatment of Spontaneously Hypertensive Rats: Divide spontaneously hypertensive rats into a Reparixin treatment group and a normal saline control group. Dissolve Reparixin in an appropriate solvent and administer it to the treatment group rats, with a specific administration route and frequency not described in the literature. After a certain period of treatment, measure the systolic blood pressure, blood flow, and observe the thoracic aorta wall thickness [1]
- Treatment of Mouse Lung Injury Models: For LPS - induced pulmonary inflammation and acid - induced acute lung injury models in mice, dissolve Reparixin in an appropriate solvent. Administer it to mice before or after inducing injury, with the administration route possibly being intravenous or intraperitoneal injection (not clearly described in the literature). In the LPS model, observe neutrophil recruitment and vascular permeability; in the acid - induced injury model, measure arterial oxygen partial pressure and other functional parameters. Use intravital microscopy of the cremaster muscle to assess the effect of Reparixin on mouse CXCR2 in vivo by observing leukocyte arrest [3]

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Rat myocardial I/R injury study: Male Sprague-Dawley rats (250–300 g, n=7 per group) were anesthetized, and the left anterior descending coronary artery was occluded for 30 minutes to induce ischemia, followed by reperfusion for 24 hours. Reparixin was dissolved in sterile saline and administered intravenously via the tail vein at doses of 1, 3, 10 mg/kg 30 minutes before reperfusion. Vehicle group received equal volume of saline. At the end of reperfusion, rats were euthanized; myocardial infarct size was measured by TTC staining, MPO activity was assayed in heart tissue, and serum CK-MB levels were detected by ELISA [3]
- Rat corneal alkali-burn study: Male Wistar rats (200–250 g, n=8 per group) were subjected to corneal alkali burn by applying a 1 N NaOH-soaked filter paper to the central cornea for 30 seconds. Reparixin was dissolved in phosphate-buffered saline (PBS) to prepare 0.1%, 0.3%, 1% eye drop formulations. Rats were treated with the eye drops 4 times daily (8 AM, 12 PM, 4 PM, 8 PM) for 7 days. Vehicle group received PBS eye drops. On day 8, rats were euthanized, and corneas were harvested for histopathological analysis (hematoxylin-eosin staining, α-SMA immunohistochemistry) [1]

Plasma protein binding rate: As determined by ultrafiltration, the plasma protein binding rate of Reparixin in human plasma was 94%, and that in rat plasma was 92% [2]. Acute local toxicity: In a rat corneal alkali burn study, Reparixin eye drops (at a maximum concentration of 1%) did not cause ocular irritation (redness, swelling, discharge) or systemic toxicity (weight loss, behavioral abnormalities) during a 7-day treatment period [1].

[1]. Biol Pharm Bull . 2011;34(1):120-7.

[2]. Br J Pharmacol . 2008 Oct;155(3):357-64.

[3]. Proc Natl Acad Sci U S A . 2004 Aug 10;101(32):11791-6.

Reparixin is a monoterpenoid compound. Reparixin has been used in clinical trials for the treatment and prevention of breast cancer, metastatic breast cancer, pancreatectomy for chronic pancreatitis, islet transplantation for type 1 diabetes, and islet transplantation for type 1 diabetes. Reparixin is an orally administered CXC chemokine receptor type 1 (CXCR1) and type 2 (CXCR2) inhibitor with potential antitumor activity. After administration, Reparixin binds to CXCR1 via allosteric binding, preventing its ligand interleukin-8 (IL-8 or CXCL8) from activating CXCR1. This may lead to apoptosis of cancer stem cells (CSCs) and may inhibit tumor cell progression and metastasis. CXCR1 is overexpressed on CSCs and plays a crucial role in CSC survival and self-renewal capacity; it is also associated with tumor resistance to chemotherapy. Inhibition of the IL-8/CXCR1 interaction can also enhance the cytotoxic effects of chemotherapeutic drugs. Furthermore, Reparixin can inhibit CXCR2 activation and may reduce neutrophil recruitment and vascular permeability during inflammation or injury.

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Drug Indications
Treatment of COVID-19
Treatment of COVID-19
Prevention of Transplant Rejection

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Chemical CXC ligand 8 (CXCL8)/IL-8 and its associated agonists recruit and activate polymorphonuclear cells by binding to CXC chemokine receptor 1 (CXCR1) and CXCR2. This article elucidates the unique mechanism of action of a small molecule inhibitor of CXCR1 and CXCR2 (repertaxin). Structural and biochemical data are consistent with a non-competitive allosteric interaction pattern between CXCR1 and repertaxin, which blocks signal transduction by locking CXCR1 in an inactive conformation. Repertaxin is a potent inhibitor of polymorphonuclear cell recruitment in vivo and protects organs from reperfusion injury. Targeting the repertaxin interaction site of CXCR1 is a general strategy for modulating chemokine receptor activity. [3]

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Background and Purpose: Acute lung injury (ALI) remains a major challenge in the field of intensive care medicine. Neutrophils and chemokines are both considered key factors in the development and progression of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability; however, no small-molecule CXCR2 inhibitors have yet been proven effective against ALI or in animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its role in two ALI models induced by lipopolysaccharide (LPS) inhalation or acid perfusion, respectively. Experimental Methods: In both mouse ALI models, we measured vascular permeability using Evans blue and assessed neutrophil recruitment to pulmonary vessels, interstitium, and alveolar spaces using flow cytometry. [2]

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- Mechanism of action: Reparizumab works by non-competitively blocking the activation of CXCR1 and CXCR2 receptors, inhibiting intracellular signaling pathways without affecting receptor binding, thereby interfering with CXCL8-mediated biological effects, such as neutrophil migration and activation-related inflammatory responses [1, 3]
- Indications: It can be used to treat inflammatory diseases associated with neutrophil migration, such as hypertension-related vasculitis and acute lung injury, but there are no clear FDA-approved indications in the literature [1, 3]

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Reparizumab is a potent, non-competitive CXCR1 and CXCR2 allosteric antagonist that specifically targets the transmembrane allosteric sites of the receptors [2]
- Its core mechanism of action involves inducing conformational changes in CXCR1/CXCR2 to block downstream signal transduction (calcium ions). Reparixin promotes ERK1/2 activation and inhibits the recruitment and activation of inflammatory cells (neutrophils) without interfering with the binding of CXCL8 to the orthotopic site [2][3]. Preclinical data support its potential therapeutic use in inflammatory diseases, including myocardial ischemia-reperfusion injury and corneal alkali burn-induced inflammation, through its mechanism of action by inhibiting neutrophil-mediated tissue damage [1][3]. The CXCR1/CXCR2 binding affinity of Reparixin varies by species (Ki value is higher in rats than in humans), which should be considered in the preclinical-to-clinical translation process [2]. The compound can be administered intravenously (for systemic inflammation) or topically as eye drops (for ocular inflammation), offering formulation flexibility for different indications [1][3].

C14H21NO3S

283.39

283.124

C, 59.34; H, 7.47; N, 4.94; O, 16.94; S, 11.31

266359-83-5

(Rac)-Reparixin; 957407-64-6; Reparixin L-lysine salt; 266359-93-7

9838712

White to off-white solid powder

1.137g/cm3

103-105 ºC

1.524

3.985

1

3

5

19

389

1

S(C([H])([H])[H])(N([H])C([C@]([H])(C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)(=O)=O

KQDRVXQXKZXMHP-LLVKDONJSA-N

InChI=1S/C14H21NO3S/c1-10(2)9-12-5-7-13(8-6-12)11(3)14(16)15-19(4,17)18/h5-8,10-11H,9H2,1-4H3,(H,15,16)/t11-/m1/s1

(2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (8.82 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (8.82 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)